There is a growing need in the biotechnology, biopharmaceutical, and research fields for a fast, efficient, versatile, and inexpensive low volume, one-step, micro-scale method, in kit form, to prepare small quantities of proteins for early-stage exploratory research. To move forward with early-stage exploratory research, it is essential to be able to quantitate the levels of the proteins of interest and to remove substances that might interfere with the properties and functions of these proteins. Protein quantitation is often accomplished via in vitro quantitative assays such as those employing optical detection methods. Such assay methods include, but are not limited to, colorimetry, fluorimetry, spectrophotometry, microtitre plate scanning, and optical microfluidics. Each of these optical assay methods suffers greatly in accuracy, producing falsely high and, on occasion, falsely low signals, if the sample in question contains unwanted, optically absorptive contaminants or if the sample is cloudy or turbid. Specific interference may occur, on a case-by-case basis, from substances that chemically react with assay reagents or that inhibit development of the assay in some fashion, as in the case of enzyme inhibitors. Thus, for accurate quantification of the protein of interest, removal of such contaminants is essential.
A single, low-volume, micro-scale, one-step method (in kit form) for wholesale contaminant reduction would be highly desirable. If applicable to a large variety of proteins from many different sources, the method would be even more desirable. Furthermore, a kit-based method that is versatile, fast, efficient, and cost-effective is even more desirable. Lastly, if that method is fully scaleable, it could have significant applications in large-scale commercial protein production. Thus, if the same micro-scale method used as a kit to pre-screen proteins were to be almost infinitely scaleable (while remaining fast, efficient, versatile, and inexpensive), then it could be applied, economically and effectively, to large-scale protein production and manufacturing. Outstanding benefits would accrue if the same one-step method could achieve, on both the micro-scale and the macro-scale, very significant reductions in levels of DNA, cell debris and other particulates, lipids, water-soluble and water-insoluble pigments and other small molecules, and many extraneous, contaminating proteins. Further benefits could arise if the same method were to greatly lower viscosity while greatly reducing total sample volume.
Such needs are especially relevant for naturally occurring proteins that have neither been cloned nor genetically engineered to contain an affinity ligand that facilitates rapid protein purification by affinity chromatography or affinity trapping methods. While cloning and affinity tagging a protein is often the method of choice for late-stage protein research, development, and manufacturing, cloning and affinity tagging is not always cost-effective or time-efficient for very early-stage proteins that are more likely than not to fail in early trials. It is much more cost-effective to perform early trials on proteins extracted from natural sources. But, as proteins from natural sources have no affinity tags, extraction, purification, viscosity reduction, particulate removal, and contaminant reduction often require multiple, tedious, and time-consuming steps. A one-step procedure for naturally occurring proteins (equivalent in many respects to affinity methods for cloned proteins) would be highly desirable. Furthermore, even in cases where a protein has succeeded in early trials and has been cloned and affinity tagged, that protein must be extracted from cells in a cost-effective way and must be prepared for affinity chromatography in ways that facilitate effective separation of the protein of interest while preserving the resolving power and the lifetime of the affinity adsorbent—often a very expensive material. Usually, such pre-column preparations involve multiple, independent steps beginning with non-selective extraction of all water-soluble components of the cells by cell lysis methods such as sonication, homogenization, freeze-thaw lysis, lysozyme treatment, bead mills, French press, or organic solvent treatment. Following extraction, it may be necessary, so as to effect resolving power of the affinity column and to increase column lifespan, to separate out particulates, to lower viscosity by removing DNA, polysaccharides, and lipid micelles, and to effect buffer exchange to facilitate proper affinity binding conditions and/or prevent precipitation of contaminants during affinity chromatography. In the case of immobilized metal affinity chromatography (IMAC), the HIS6-tagged recombinant protein cannot be successfully trapped by the immobilized metal ion without prior removal of other chelators such as citrate, ammonia, and EDTA. Thus a buffer exchange is often required to remove such materials and to create a slightly alkaline pH environment.
Examples of naturally occurring proteins for which a cost-effective and time-efficient purification method is needed include peroxidases, antibodies, fluorescent proteins such as GFP, and other proteins commonly employed in research, diagnostics and therapeutics. For example, horseradish peroxidase (HRP), a protein of approximately 40 kDa, is one of the two most widely used enzyme labels in medical diagnostics and research applications, the other being alkaline phosphatase. Horseradish peroxidase is applied often in immunoassays and nucleic acid hybridization assays, in part because of the availability of peroxidase-conjugated antibodies to haptens such as biotin, fluorescein isothiocyanate, and digoxigenin.
Horseradish peroxidase has been isolated using various methods. For example, Shannon et al. ((1966) J. Biol. Chem. 241:2166) teach ammonium sulfate fractionation followed by CM-cellulose and DEAE-cellulose chromatography. Keilin and Hartree ((1951) Biochem. J. 49:88) teach ammonium sulfate fractionation, precipitation with ethanol, fractionation with calcium phosphate and ethanol, and fractionation with ammonium sulfate. Kenten and Mann ((1954) J. Biochem. 57:347-348) disclose purification of horseradish peroxidase by ethanol:chloroform extraction followed by ammonium sulfate fractionation and ethanol precipitation.
However, there is a need in the art for improved methods for high yield purification of proteins and enzymes. The present invention meets this need in the art.